In vitro broad-spectrum antiviral activity of MIT-001, a mitochondria-targeted reactive oxygen species scavenger, against severe acute respiratory syndrome coronavirus 2 and multiple zoonotic viruses

Highlights • MIT-001 demonstrates potent antiviral activity against SARS-CoV-2 and zoonotic viruses, offering a promising therapeutic approach for viral infections.• MIT-001 inhibits the replication of SARS-CoV-2 variants, ZIKV, SEOV, and VACV, indicating broad-spectrum antiviral efficacy.• Restoration of antioxidant gene expression highlights MIT-001 ability to counteract oxidative stress and enhance cellular defense mechanisms compromised by SARS-CoV-2 infection.• MIT-001 preserves mitochondrial function and cellular homeostasis by mitigating mitochondrial depolarization caused by SARS-CoV-2 infection.


Introduction
Coronavirus Disease 2019 (COVID-19) began in Wuhan, Hubei province, China, in 2019.On 30 January 2020, the World Health Organization (WHO) declared a global emergency over the novel coronavirus outbreak (Sohrabi, 2020).Subsequently, on 11 March 2020, the WHO declared COVID-19 a pandemic (Cucinotta, 2020).According to the WHO Health Emergency Dashboard, by May 2023, approximately 767 million confirmed cases and over 6.9 million deaths had been reported (WHO Health Emergency Dashboard).COVID-19 is an infectious disease caused by the severe acute respiratory syndrome 2 virus (SAR-S-CoV-2).The outbreak of COVID-19 has severely impacted the global economy and human health.
In the quest for effective antiviral treatments against COVID-19, recent studies have focused on viral genetic components and host genetic factors as main potential targets.Molnupiravir, a nucleotide analog inducing replication errors, is an approved antiviral agent which is disrupting viral replication, whereas the combination therapy of Nirmatrelvir and Ritonavir targets the viral protease (Mpro) against SARS-CoV-2 (Saravolatz et al., 2023;Lamb, 2022).Simultaneously, host factors regulating viral susceptibility and immune response pose a critical target for developing antiviral countermeasures.4-Octyl itaconate (4-OI), activating the Nuclear factor erythroid-2-related factor 2 (Nrf2)-related pathway, enhanced antioxidant defenses and inhibited pro-inflammatory responses upon viral infections, holding potential for antiviral effects against SARS-CoV-2 (Olagnier et al., 2020).These multifaceted strategies demonstrate the diverse approaches being pursued to develop effective antiviral therapies against COVID-19 and multiple viral outbreaks.
Here, we introduce a potential mitochondria-targeting anti-inflammatory and anti-reactive oxygen species (ROS) agent, MIT-001 (Known as NecroX-7) against SARS-CoV-2 and multiple viruses in vitro.MIT-001 inhibits ROS and calcium accumulation in mitochondria, which suppresses the release of damage-associated molecules by accidental necrosis and the agent-induced antioxidant action of nuclear factor kappalight-chain-enhancer of activated B cells (NF-кB) and inflammasomedependent cytokines (Grootaert et al., 2016;Im et al., 2015).Currently, the antiviral activity of MIT-001 remains to be investigated.
In this study, the antiviral activity of MIT-001 was evaluated against SARS-CoV-2 in vitro.In addition, we examined the efficacy of MIT-001 in DNA and RNA viruses to verify its broad-spectrum antiviral activity.MIT-001 could serve as a broad-spectrum antiviral agent for the treatment of SARS-CoV-2 and variants (B.1.617.2 and BA.1 strains), Seoul virus (SEOV), Zika virus (ZIKV), and Vaccinia virus (VACV).

Ethics
The study of multiple anti-viral reagents against SARS-CoV-2 was performed at biosafety level-3 (facilities at Hallym Clinical and Translational Science Institute, Hallym University, Chuncheon, South Korea) under guidelines and protocols in line with the institutional biosafety requirements (Hallym2020-04, 30th, Oct., 2020, Hallym University Institutional Biosafety Committee).Experiments using ZIKV, VACV, and SEOV were performed at biosafety level-2.

RNA extraction and reverse transcription-polymerase chain reaction (RT-PCR)
Total RNA was extracted from cells using TRIzol (15596026, Ambion, Life Technologies, Carlsbad, CA, USA) according to the manufacturer's protocol.Subsequently, the extracted RNA was subjected to reverse transcription to complementary DNA (cDNA) using a highcapacity RNA-to-cDNA kit (4387406, Thermo Fisher Scientific Baltics UAB) and a SimpliAmp Thermal Cycler (A24811, Thermo Fisher Scientific).The reverse transcription reaction was performed at 37 • C for minutes, followed by a denaturation step at 95 • C for 5 min.

Real-time quantitative PCR (RT-qPCR)
Quantification of specific viral RNA was performed using Power SYBR® Green PCR Master Mix (4367659, Applied Biosystems™, Life Technologies Ltd., Woolston Warnington, UK) and QuantStudio3 Real-Time PCR instrument (A28132, Applied Biosystems™).The primer list is shown in Tables 1 and 2.

Plaque assay
Vero E6 cells (1 × 10 6 cells per well) were seeded into 6-well plates and incubated overnight at 37 • C in 5 % CO 2 .Cells were washed with PBS and infected with serial dilutions of SARS-CoV-2 supernatant using serum-free medium for 90 minutes.The plates were shaken once every 15-20 min for virus adsorption.Overlay medium (DMEM/F12 medium) containing 0.6 % agar was added and incubated at 37 • C with 5 % CO for 4 days.Formaldehyde was used for fixation and crystal violet staining was performed.

Total RNA-sequencing (Total RNAseq) analysis
TruSeq Stranded Total RNA Library Prep Gold Kit (Illumina, San Diego, CA, USA) was used for library preparation of total RNA sequencing data.The cDNA fragments obtained through RNA sequencing analysis were mapped to the genomic reference (GRCh38) using the HISAT2 program which utilizes the Bowtie2 aligner for spliced read mapping (Kim, Daehwan et al., 2019;Langmead et al., 2012).The processed and mapped reads for each sample were quantified and known genes/transcripts were assembled using the StringTie program with a reference gene model (Pertea et al., 2015).The abundance of transcripts was calculated as read count and normalized using fragments per kilobase of transcript per million mapped reads (FPKM) and transcripts per kilobase million (TPM) values.Differential gene expression analysis (DEG) was performed using the read count values, applying the StringTie-e option for original raw data, filtering genes with low quality, and using the edge R library-calcNormFactors to calculate TMM (Trimmed mean of M-values) normalization (adjusted p-value < 0.05; derived from a hypergeometric test & multiple testing correction (FDR) and fold change |Fc| ≥ 2).

Mitochondrial membrane potential staining
The mitochondrial membrane potential probe JC-1 (T3168, Thermo Fisher Scientific) was used to detect the recovery of mitochondrial membrane potential.JC-1 is expressed as a green, fluorescent monomer (~529 nM) at depolarized and abnormal mitochondrial membrane potentials.In mitochondria with a normally functioning proton pump and normal and hyperpolarized membranes, JC-1 is concentrated inside the mitochondria and forms red fluorescent J aggregates (~590 nM).To stain hACE2-A549 cells seeded on the cover glass in a 12-well plate, JC-1 was diluted to 10 mM and used at a final concentration of 2 µM in serum free DMEM for 20 min at 37 • C with 5 % CO 2 .Control cells were treated with H 2 O 2 for 20 min.After JC-1 staining, Hoechst 33342 (62249, Thermo Fisher Scientific) was used to stain the nucleus for 5 min at RT.After each staining step, cells were washed three times using prewarmed PBS and mounted on the slide glass.All steps were carried out with the light blocked.

Cell counting kit-8 assay
Cell viability was determined using Cell Counting Kit-8 (CCK8) assay (Dojindo Molecular Technologies, Kumamoto, Japan) following the manufacturer's protocol.Briefly, cells were plated in 96-well plates (10 4 cells/200 μL /well) and incubated for 24 h before drug treatment.On next day, cells were treated with different concentrations of MIT-001 for 48 h.The plates were incubated at 37 • C and 5 % CO 2 .At 45 h post treatment, 10 μL of CCK-8 reagent was added to the wells.After 3hrs, the optical density (OD) was measured using a microplate reader at a wavelength of 450 nm.

MIT-001 inhibits the expression of pro-inflammatory genes but increases the expression of anti-oxidative genes upon SARS-CoV-2 B.1 in hACE2-A549 cells
We evaluated the effect of MIT-001 treatment on inflammatory cytokines and Nrf2-related genes in hACE2-A549 cells infected with SARS-CoV-2 B.1.The total RNAseq analysis showed that SARS-CoV-2 B.1 infection highly induced the interferon (IFN) signaling pathway and proinflammatory cytokine genes.However, MIT-001 treatment inhibited the expression of IFN signaling and pro-inflammatory cytokines upon SARS-CoV-2 B.1 infection.(Fig. 2A).Regarding Nrf2-related genes, Nrf2 signaling was initially downregulated in SARS-CoV-2 B.1-infected cells; however, MIT-001 treatment led to the upregulation of the Nrf2 pathway.In addition to the upregulation of the heme oxygenase-1 (HMOX1) due to MIT-001 treatment, our findings revealed that the restoration of biliverdin (BLVR), a heme metabolite, at the RNA level occurred (Fig. 2B).There was no significant difference in the fold change

Table 2
Primer sequences of human-related genes for RT-qPCR. of genes including interferon related genes, pro-inflammatory cytokines and Nrf2 driven genes in only MIT-001 treated cells compared with nontreated cells (not shown).RT-qPCR results confirmed the downregulation of IFNB, interferon induced protein with tetratricopeptide repeats 1 (IFIT1), IFIT2, Interleukin 6 (IL6) and tumor necrosis factor alpha (TNFA) expression after MIT-001 treatment (Fig. 2C).RT-qPCR analysis showed that the expression of HMOX1 and NAD(P)H: quinone oxidoreductase 1 (NqO1), Nrf2-induced genes associated with antioxidant activity, significantly increased after MIT-001 treatment, approaching or surpassing the levels in untreated infected cells (Fig. 2D).These results suggest that MIT-001 induces strong antiinflammatory and anti-oxidant responses in SARS-CoV-2 infected hACE2-A549 cells.

MIT-001 rescues mitochondrial membrane potential (ΔΨ m) and dynamics of SARS-CoV-2-infected hACE2-A549 cells
To evaluate the homeostasis of mitochondria after MIT-001 treatment, hACE2-A549 cells were infected with the SARS-CoV-2 B.1 at MOI of 0.1 pfu per cell.We performed JC-1 staining to detect dynamic changes in the mitochondrial membrane potential (ΔΨm).The ΔΨm (JC-1 aggregate, Red) of hACE2-A549 cells decreased after SARS-CoV-2 infection, and MIT-001 treatment of infected cells resulted in the same or higher intensity as that of the uninfected cells at 24 hpi (Fig. 3A and  B).This suggests that mitochondria are under constant stress due to SARS-CoV-2 infection and mitochondrial stress is inhibited by MIT-001 treatment.
Viral infections increase ROS activity, leading to oxidative stress and the activation of cytoprotective genes through Nrf2-dependent pathways (Deramaudt et al., 2013;Johnson et al., 2008;Herengt et al., 2021;Osburn et al., 2008;Hayes et al., 2014).However, in recent studies, SARS-CoV-2 infection enabled to inhibit the Nrf2 pathway, as observed in biopsies from COVID-19 patients (Olagnier et al., 2020;Zhang et al., 2022a;Zhang et al., 2022b).The SARS-CoV-2 non-structural protein 14 (nsp14) interacted with Sirtuin 1 (SIRT1) to inhibit the activation of Nrf2 pathway (Fratta et al., 2021).The treatment of 4-OI has been shown to activate the Nrf2 pathway in SARS-CoV-2 infected cells by dissociating the Keap1-Nrf2 complex in the cytoplasm.This activation of Nrf2 led to the inhibition of SARS-CoV-2 replication and subsequent pro-inflammatory responses.The transcription factor Nrf2 regulates the expression of antioxidant genes such as HMOX1 and NqO1 (Maines et al., 2005;Ma et al., 2013;Tonelli et al., 2018).Previous studies have shown that HMOX1-induced BLVR mitigated the replication of SARS-CoV-2 and Ebola virus (EBOV) (Olagnier et al. 2020;Fratta et al. 2021;De Clercq et al. 2015).Our study demonstrated that treatment with MIT-001 effectively restored the decreased expression of HMOX1 caused by SARS-CoV-2 infection to levels comparable to those observed in uninfected cells.Furthermore, the restoration of HMOX1 gene expression resulted in the up-regulation of BLVR A and B, which may potentially influence the antiviral activity of MIT-001 against the virus.However, further investigations are required to elucidate the precise mechanism of action underlying the therapeutic effects of MIT-001.
The study has limitations: Firstly, the antiviral activity of MIT-001 was only tested in vitro, requiring further evaluation in animal models and humans to assess safety and efficacy.Secondly, a limited number of viruses were tested, necessitating additional research to determine the antiviral activity of MIT-001 against other viruses.Thirdly, the mode of action remains to be further investigated.
In conclusion, MIT-001 provides broad-spectrum antiviral activity against SARS-CoV-2 and multiple zoonotic viruses in vitro.This study highlights the potential of MIT-001 for developing a novel mitochondria-targeted antiviral against emerging viral infections.

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Table 3
Mean of EC 50 of MIT-001 against different strains of SARS-CoV-2 and multiple viruses.T.Lim et al.antiviral activity against SARS-CoV-2 and various zoonotic viruses, providing a promising therapeutic strategy for combating viral infections.The efficacy of MIT-001 extends to inhibiting the replication of SARS-CoV-2 variants, ZIKV, SEOV, and VACV.Moreover, MIT-001 demonstrates the ability to restore the expression of key antioxidant genes HMOX1 and NqO1, which are typically reduced by SARS-CoV-2 infection.This feature underscores MIT-001 capacity to counteract oxidative stress and enhance cellular defense mechanisms.Additionally, MIT-001 mitigates mitochondrial depolarization induced by SARS-CoV-2 infection, indicating a role in preserving mitochondrial function and cellular homeostasis during viral infection.Collectively, these findings emphasize the significant potential of MIT-001 as a promising candidate for the development of comprehensive and targeted antiviral therapies, particularly against emerging SARS-CoV-2 variants and zoonotic viral infections.